Nucleic acid sequence analysis from single cells

ABSTRACT

Presented herein are methods and compositions for multiplexed single cell gene expression analysis. Some methods and compositions include the use of droplets and/or beads bearing unique barcodes such as unique molecular barcodes (UMI).

RELATED APPLICATIONS

This application claims priority to U.S. provisional application62/211,597 filed on Aug. 28, 2015 and is a continuation-in part of PCTapplication PCT/US15/28062, filed on Apr. 28, 2015 which claims priorityto U.S. provisional application Nos. 61/985,983 filed on Apr. 29, 2014and 61/987,433 filed on May 1, 2014. Each of these earlier filedapplications are hereby incorporated by reference in its entirety.

GOVERNMENT SUPPORT

This invention was made with government support under NationalInstitutes of Health (NIH) grant number MH098977 awarded by the PublicHealth Service (PHS). The government has certain rights in theinvention.

BACKGROUND

The determination of the mRNA content of a cell or tissue (i.e. “geneexpression profiling”) provides a method for the functional analysis ofnormal and diseased tissues and organs. Gene expression profiling isusually performed by isolating mRNA from tissue samples and subjectingthis mRNA to microarray hybridization. However, such methods only allowpreviously known genes to be analyzed, and cannot be used to analyzealternative splicing, promoters and polyadenylation signals.Additionally, microarrays have two major shortcomings: they are linkedto known genes, and they have limited sensitivity and dynamic range.

Direct sequencing of all, or parts, of the mRNA content of a tissue isbeing increasingly used. However, current methods of analyzing the mRNAcontent of cells by direct sequencing rely on analyzing bulk mRNAobtained from tissue samples typically containing millions of cells.This means that much of the functional information present in singlecells is lost or blurred when gene expression is analyzed in bulk mRNA.In addition, dynamic processes, such as the cell cycle, cannot beobserved in population averages. Similarly, distinct cell types in acomplex tissue (e.g. the brain) can only be studied if cells areanalyzed individually.

There are often no suitable cell-surface markers to use in isolatingsingle cells for study, and even when there are, a small number ofsingle cells are not sufficient to capture the range of naturalvariation in gene expression. What is needed is a method of preparingcDNA libraries which can be used to analyze gene expression in aplurality of single cells.

SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as an ASCII fileentitled IP-1388-US SL.txt, created Oct. 12, 2015, which is 6,742 bytesin size. The information in the electronic format of the SequenceListing is incorporated herein by reference in its entirety.

SUMMARY

Presented herein are methods and compositions for nucleic acid analysisfrom single cells and/or nucleic acid from single cell nuclei andorganelles. Some methods and compositions can be used for multiplexedsingle cell gene expression analysis. Some methods and compositionsinclude the use of droplets and/or beads bearing unique barcodes such asunique molecular barcodes (UMI).

In one embodiment, presented herein are methods and compositions ofnucleic acid sequence analysis from a single cell. In some embodiments,the methods and compositions of nucleic acid sequence analysis can beused to prepare sequencing library from nucleic acid from a singleorganelle. In some embodiments, the organelle can be a nuclei from asingle cell. In some embodiments, the single organelle is obtained fromsingle cell. Other exemplary organelles include but are not limited tomitochondria and ribosome.

In one embodiment, the methods include releasing nuclei from cells toprovide plurality of nuclei, wherein each nucleus is from a single cell.The nuclei are spatially separated from each other such that one nucleusis present at a spatial compartment. A first strand of cDNA issynthesized from the mRNA in each individual mRNA sample with a firststrand synthesis primer. In some embodiments, the first strand synthesisprimer is an oligo dT primer further comprising a first amplificationprimer binding site. In some embodiments, the first strand synthesisprimer is a randomer. In some embodiments, the first strand synthesisprimer is a randomer further comprising a first amplification primerbinding site. In some embodiments, the first strand synthesis primer isa mixture of oligo dT primer and randomer each further comprising afirst amplification primer binding site. In some embodiments, the methodfurther includes incorporating a template switching oligonucleotideprimer (TSO primer) along with the mixture of oligo dT primer andrandomer, each of oligo dT primer and randomer further comprising afirst amplification primer binding site. In some embodiments, the TSOprimer further comprises a second amplification primer binding site. Insome embodiments, the first strand synthesis primer is extended beyondthe mRNA template and further copies the TSO primer strand. In someembodiments, the second strand of cDNA is synthesized using the TSOprimer. In some embodiments, the second strand of cDNA is synthesizedusing the second amplification primer complimentary to the first strandof cDNA that is extended beyond the mRNA template to encompass thecomplimentary TSO strand. In some embodiments, the double stranded cDNAis amplified with first and second amplification primers. In someembodiments, the first, second or both first and second amplificationprimers are immobilized on a solid support. Exemplary solid supportsinclude beads, flow cells, microwells

In some embodiments, the double stranded cDNA is subjected totagmentation reaction such that barcodes can be introduced into thedouble stranded cDNA. Exemplary methods of tagmentation are disclosed inU.S. Pat. Nos. 9,115,396; 9,080,211; 9,040,256; U.S. patent applicationpublication 2014/0194324. Each of which is incorporated herein byreference in its entirety. In some embodiments, the barcodes can be usedto determine the contiguity information of the sequence. In someembodiments, the barcodes can be used as a source identifier.

In some embodiments, the method includes incorporating a tag into thecDNA to provide a plurality of tagged cDNA samples, wherein the cDNA ineach tagged cDNA sample is complementary to mRNA from a single cell. Inone embodiment, the tag comprises a cell-specific identifier sequenceand a unique molecular identifier (UMI) sequence. In some embodiments,the first strand synthesis primer comprises a tag. In some embodiments,the TSO primer comprises a tag. In some embodiments, tagged cDNA fromthe nuclei of single cells can be pooled and optionally amplified.

In some embodiments, the methods include preparing sequencing libraryfrom microRNA (miRNA), small interfering RNA (siRNA), ribosomal RNA, ormitochondrial DNA from a single cell. The method includes releasing thecell organelles from a single cell to provide plurality of organelles.The organelles are spatially separated from each other such that oneorganelle is present at a spatial compartment. A first strand of DNA issynthesized from the microRNA (miRNA), small interfering RNA (siRNA),ribosomal RNA, or mitochondrial DNA with a first strand synthesisprimer. In some embodiments, the first strand synthesis primer is arandomer. In some embodiments, the first strand synthesis primer is arandomer further comprising a primer binding site. In some embodiments,the first strand synthesis primer is a mixture of a first strandspecific synthesis primer and a randomer. In some embodiments, the firststrand synthesis primer is a mixture of a first strand specificsynthesis primer and a randomer, each further comprising a firstamplification primer binding site.

In some embodiments, the primer that binds to the first primer bindingsite is an amplification primer. In some embodiments, the method furtherincludes incorporating a template switching oligonucleotide primer (TSOprimer) along with the mixture of a first strand specific synthesisprimer and a randomer, each of first strand specific synthesis primerand a randomer further comprising a first primer binding site. In someembodiments, the TSO primer further comprises a second primer bindingsite. In some embodiments, the primer that binds to the second primerbinding site is an amplification primer. In some embodiments, the firststrand synthesis primer is extended beyond the microRNA (miRNA), smallinterfering RNA (siRNA), ribosomal RNA, or mitochondrial DNA templateand further copies the TSO primer strand. In some embodiments, thesecond strand of DNA is synthesized using the TSO primer. In someembodiments, the second strand of DNA is synthesized using the secondprimer complimentary to the first strand of DNA that is extended beyondthe template RNA or DNA to encompass the complimentary TSO strand. Insome embodiments, the double stranded DNA is amplified with first andsecond primers.

In one embodiment, the organelles such as nuclei, mitochondria,ribosomes are spatially separated by fluorescence activated cell sorting(FACS) and each organelle is sorted into a spatial compartment, e.g.,single microwell on a Fluidigm C1 chip. In some embodiments, eachorganelle is spatially separated into a spatial compartment by beingimmobilized on a solid surface. For example, through an antibody,wherein the antibody specifically binds to the organelle and theantibody is immobilized on a solid surface. In some embodiments, thesolid surface is a flow cell or a bead.

In some embodiments, the randomers comprise one or more quasi-randomprimers that are selected from the group consisting of an AT-rich set ofrandom amplification primers; a set of random amplification primerscomprising AT-rich 5′ termini; a set of variable-length randomamplification primers, wherein each primer comprises a random 3′ portionand a degenerate 5′ terminus, the degenerate 5′ terminus of which can beproportional in length to the A/T content of the random 3′ portion ofthe primer; a set of Tm-normalized random primers, wherein each primerof the set comprises one or more base analogues that can normalize theTm of each primer to the Tm of other primers in the set of primers; aset of random primers, wherein each primer comprises a random 3′ portionand a constant 5′ priming portion; a set of random amplificationprimers, wherein each primer comprises a random 3′ portion and aconstant 5′ priming portion, and wherein the random 3′ portion comprisesRNA; a set of random amplification primers, wherein each primercomprises a random 3′ portion and a constant 5′ priming portion, andwherein the random 3′ portion comprises at least one non-natural baseselected from the group consisting of nucleic acids including2′-deoxy-2-thiothymidine (2-thio-dT), 2-aminopurine-2′-deoxyriboside(2-amino-dA), N4-ethyl-2′-deoxycytidine (N4-Et-dC), N4-methyldeoxycytidine (N4-Me-dC), 2′-deoxyinosine, 7-deazaguanine (7-deaza-G),7-iodo-7-deazaguanine (I-deazaG), 7-methyl-7-deazaguanine, (MecG),7-ethyl-7-deazaguanine (EtcG) and any combination of the foregoing setsof primers. The quasi-random primers set forth above are described infurther detail herein. In some embodiments described herein, thequasi-random primers are provided in pairs or sets.

One embodiment presented herein is a method of preparing a cDNA libraryfrom a plurality of single cells, the method comprising the steps of:releasing mRNA from each single cell to provide a plurality ofindividual mRNA samples, wherein the mRNA in each individual mRNA sampleis from a single cell; synthesizing a first strand of cDNA from the mRNAin each individual mRNA sample with a first strand synthesis primer andincorporating a tag into the cDNA to provide a plurality of tagged cDNAsamples, wherein the cDNA in each tagged cDNA sample is complementary tomRNA from a single cell. In one embodiment, the tag comprises acell-specific identifier sequence and a unique molecular identifier(UMI) sequence. In some embodiments, the tag comprises a cell-specificidentifier sequence without the UMI. The method further comprisespooling the tagged cDNA samples; optionally amplifying the pooled cDNAsamples to generate a cDNA library comprising double-stranded cDNA; andperforming a tagmentation reaction to simultaneously cleave each cDNAand incorporate an adapter into each strand of the cDNA, therebygenerating a plurality of tagged cDNA fragments. In some embodiments,sufficient number of single cells is present, the amplification of cDNAcan be avoided. The second strand of cDNA is synthesized using atemplate switching oligonucleotide primer (TSO primer), followed bysymmetric Nextera.

In certain embodiments, the method further comprises amplifying thetagged cDNA fragments to generate amplified tagged cDNA fragments. Insome aspects, amplifying comprises adding additional sequence to the 5′end of the amplification products.

In some aspects, the additional sequence comprises primer bindingsequence for amplification on a solid support. In some aspects, theadditional sequence comprises additional index sequences.

In certain embodiments, the method further comprises amplifying theamplified tagged cDNA fragments on a solid support.

In certain embodiments, the method further comprises sequencing theamplification products on the solid support.

In some aspects, the tagmentation reaction comprises contacting thedouble-stranded cDNA with a transposase mixture comprising adaptersequences that are not found in the first strand synthesis primer.

In some aspects, the transposase mixture consists essentially oftransposomes having one type of adapter sequence.

In certain embodiments, the method further comprises sequencing thetagged cDNA fragments.

In some aspects, sequencing comprises 3′ tag counting.

In some aspects, sequencing comprises whole transcriptome analysis.

In some aspects, first strand synthesis is performed using a mixture ofrandom primers, the random primers further comprising a tag.

In some aspects, the first strand synthesis primer comprises adouble-stranded portion. In some embodiments, the first strand synthesisprimer comprising a double-stranded portion further comprises a singlestranded loop at one end. In some aspects, the first strand synthesisprimer comprises a region capable of forming a hairpin.

In some aspects, the first strand synthesis primer reduces concatenationbyproducts compared to a single-stranded first strand synthesis primer.

In some aspects, the first strand synthesis primer comprises a region ofRNA.

In some aspects, the first strand synthesis primer is hybridized to acomplementary oligonucleotide, thereby forming a double strandedportion.

Also presented herein is a plurality of beads, wherein each beadcomprises a plurality of oligonucleotides, each oligonucleotidecomprising: (a) a linker; (b) an amplification primer binding site; (c)optionally a Unique Molecular Identifier which differs for eacholigonucleotide; (d) a bead-specific sequence that is the same on eacholigonucleotide on the bead but is different on other beads; and (e) acapture sequence for capturing mRNAs and priming reverse transcription.

In some aspects, the capture sequence comprises oligo-dT.

In some aspects, each bead is in a separate droplet segregated fromother beads.

The details of one or more embodiments are set forth in the accompanyingdrawings and the description below. Other features, objects, andadvantages will be apparent from the description and drawings, and fromthe claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an exemplary schematic showing a schematic of cDNA synthesisusing SMARTer methodology.

FIG. 2A is an exemplary schematic showing first strand synthesisaccording to one embodiment, primed with oligo dT that has been appendedwith an optional molecular barcode (UMI), a sample barcode (BC), anamplification primer binding sequence (V2.A14) and a template switch(TS) primer sequence, followed by template switching, pooling of samplesand single primer PCR. Figure discloses SEQ ID NOS 14, 14-15, 14-17, and16, respectively, in order of appearance.

FIG. 2B is an exemplary schematic showing tagmentation of pooledamplification products with symmetric Nextera, followed by amplificationusing different forward (V2.615) and reverse (V2.A14) PCR primers andsubsequent paired end sequencing. Figure discloses SEQ ID NOS 17, 16,17, 16, 17, 16, 17, and 16, respectively, in order of appearance.

FIG. 3A is an exemplary schematic first strand synthesis according toone embodiment, primed with oligo dT that has been appended with asample barcode (BC), an optional molecular barcode (UMI), anamplification primer binding sequence (V2.A14) and a template switch(TS) primer sequence, followed by template switching, pooling of samplesand single primer PCR. The optional UMI is at the 5′-end of the BC.Figure discloses SEQ ID NOS 14, 14-15, and 14-16, respectively, in orderof appearance.

FIG. 3B is an exemplary schematic showing tagmentation of pooledamplification products, amplification and sequencing according to oneembodiment. Figure discloses SEQ ID NOS 16, 18, 16, 18, 16, 18, 16, 18,16, 18, and 16, respectively, in order of appearance.

FIG. 4A is an exemplary schematic showing first strand synthesisaccording to one embodiment. Figure discloses SEQ ID NOS 14 14-15, and14-16, respectively, in order of appearance.

FIG. 4B is an exemplary schematic showing tagmentation of pooledamplification products, amplification and sequencing according to oneembodiment. Figure discloses SEQ ID NOS 16, 18, 16, 18, 16, 18, 16, 18,16, 18, and 16, respectively, in order of appearance.

FIG. 5 is an exemplary schematic showing a method of pooling andsequencing multiplexed samples according to one embodiment.

FIG. 6 is a table comparing methods of gene expression analysis using100 pg human brain reference RNA.

FIG. 7 is a table comparing methods of high throughput single cell geneexpression analysis using single cells.

FIG. 8 shows graphs comparing transcript coverage with tagmentationusing only one transposase adaptor (V2.B15) versus tagmentation usingstandard/asymmetric tagmentation, using two transposase adaptors (V2.A14and V2.B15).

FIG. 9A is a schematic showing whole transcriptome analysis entailingfirst strand synthesis using randomers, according to one embodiment.Figure discloses SEQ ID NOS 14 and 19, respectively, in order ofappearance.

FIG. 9B is a schematic showing concatenation byproducts that may beproduced when using randomers for first strand synthesis.

FIG. 10 shows various primer designs to reduce or avoid concatenationbyproducts.

FIG. 11 is a schematic showing droplet-based barcoding according to oneembodiment.

FIG. 12 is a schematic showing bead-based barcoding according to oneembodiment. Figure discloses SEQ ID NOS 16, 20, 16, and 21,respectively, in order of appearance.

FIG. 13 is a schematic showing the single nuclei RNA-Sequencingworkflow.

FIG. 14 shows the steps of cDNA synthesis from single nuclei.

FIG. 15 is a schematic showing cDNA synthesis using the template switchSMARTer method. Figure discloses SEQ ID NOS 14, 14, 22, 14, and 22,respectively, in order of appearance.

FIG. 16 is a schematic of cDNA synthesis was performed in the presenceof a random primer in addition to the oligodT primer. Figure disclosesSEQ ID NOS 14 and 22, respectively, in order of appearance.

FIG. 17 shows the advantages if the SMART-Plus assay method. FIG. 17Ashows the assay performance of SMART-plus assay compiled from more than1000 high quality single nuclei. FIG. 17B shows the transcript coveragedata.

FIG. 18 shows the improved assay sensitivity of the SMART-Seq Plus overthe SMART-seq method.

DETAILED DESCRIPTION

Presented herein are methods and compositions for multiplexed singlecell gene expression analysis. Some methods and compositions include theuse of droplets and/or beads bearing unique barcodes such as uniquemolecular barcodes (UMI).

Currently the most commonly used method for single cell RNA-Seq is basedon CLONTECH™ SMART-SEQ™ technology or derivatives thereof. In short, anoligo(dT) primer primes the first-strand cDNA synthesis reaction. Whenthe reverse transcriptase (SMARTSCRIBE™) reaches the 5′ end of the mRNA,the enzyme's terminal transferase activity adds a few additionalnon-template nucleotides to the 3′ end of the cDNA. A template-switcholigo, designed to base-pair with this non-template nucleotide stretch,anneals and creates an extended template to enable the RT continuereplicating to the end of the oligonucleotide (FIG. 1).

The methods presented herein can include methods of generating taggedcDNA with sample-specific tags as described, for example, in thedisclosure of U.S. 2012/0010091, the content of which is incorporatedherein by reference in its entirety. As used herein, the termssingle-cell tagged reverse transcription and STRT refer to methodsdisclosed, for example in the incorporated materials of U.S.2012/0010091. In some embodiments, the double stranded cDNA is notdegraded with DNase in the STRT method. Instead, the double strandedcDNA is tagmented with a transposase, for example, standard Nextera.

The double stranded cDNA can then be converted into a sequencing libraryusing for example NEXTERA™ or TRUSEQ™ (Illumina, Inc.) for wholetranscriptome RNA-Seq; or through enzymatic degradation, such as DNase Ior Fragmentase, followed by adaptor ligation for 5′ end sequencing. Bothmethods have pros and cons: the former can only be multiplexed aftersample barcodes have been introduced during the library prep whereas thelatter can be multiplexed after cDNA synthesis as barcodes can beintroduced during the 1st strand synthesis step. Therefore higherthroughput and lower cost per sample favor the latter. However, theinformation obtained with both methods has different applications: theformer allows for sequencing of the whole transcriptome whereas thelatter interrogates gene expression levels only.

Herein are presented rapid gene expression library preparation methodsthat can be applied to single cell input levels and that allows for highlevels of sample multiplexing early on in the protocol.

In some embodiments, first strand synthesis is primed with an (anchored)oligo dT primer (or potentially with a randomer or a combination of thetwo) that is appended with a sample barcode (BC), an amplificationprimer binding site, and optionally a template switch (TS) primersequence. In some embodiments, the amplification primer binding site isa transposase adapter sequence, such as, for example, the Nexteraadaptor sequence V2.A14 or V2.B15. The barcode can be preceded orfollowed by a molecular barcode (unique molecular identifier, or “UMI”)that would allow for the detection of PCR duplicates. When the reversetranscriptase reaches the 5′ end of the mRNA, template switch occurs asdescribed above and in the incorporated materials of U.S. 2012/0010091.This incorporates the complement of the TS primer sequence into the 1ststrand cDNA. Because a sample barcode has been introduced into the 1ststrand cDNA, different samples can at this point be pooled. The 1ststrand pool will subsequently be rendered double stranded and optionallyamplified in a PCR reaction with the TS primer (FIG. 2A). Due to thefact that both ends of the cDNA contain complementary sequences,formation of hairpin structures will result in suppression ofamplification of smaller fragments such as artifacts, etc.

In some embodiments, as set forth in FIGS. 2A and 3A, 1st strandsynthesis is primed with an oligo dT that has been appended with asample barcode (BC), a copy of a transposase adaptor sequence the(“Nextera V2.A14 sequence”), optionally with a molecular barcode (UMI),and the template switch (TS) primer sequence. Template switch at the 3′end of the cDNA strand incorporates TS' primer sequence at the other endof the 1st strand. cDNAs of different samples can be pooled at thispoint.

In some embodiments, as set forth in FIGS. 2B and 3B, the cDNAs areamplified with a TS oligo. This single primer PCR will suppress smallamplicons such as primer dimer, etc. The cDNA pool can be tagmented witha transposase, for example, (“NEXTERA™”) that only contains one adaptorsequence, rather than the typical two adapters. In the example shown inFIGS. 2B and 3B, transposases are loaded with V2.B15 oligos. Aftertagmentation, PCR with p5-V2.A14 and p7-V2.B15 amplification primerspreferentially amplifies the 3′ end fragments of the cDNA. Fragmentsthat were generated by two tagmentation events (“symmetric fragments”)will be suppressed during the PCR and will not generate sequenceablefragments. For example, as shown in FIGS. 2B and 3B, amplificationproducts generated from symmetric fragments will present P7 primersequence at both the 5′ and 3′ end of the amplification product, andwill not form sequenceable clusters on a standard Illumina flowcellbearing P5 and p7 amplification primers. Additionally, they will besuppressed during PCR due to their complementary ends. In contrast, the3′ terminal fragment will bear P5 and P7 primer binding sites afteramplification, and can form sequenceable clusters on an Illuminaflowcell. Paired end sequencing will result in the sample BC and UMIsequence during read 1 and cDNA sequence during read 2.

Accordingly, in certain embodiments presented herein, rather thanperforming sequencing of the entire transcript, a form of a digital geneexpression assay is performed that relies on 3′ tag counting. Themethods presented herein offer the ability to individually barcode cellsat the first strand synthesis step. Additionally, barcoding of the 3′end of cDNA with inexpensive oligo-dT primers or randomers providessignificant cost savings advantages. Furthermore, use of randomers forcDNA synthesis is advantageous because it makes the method more similarto total-RNA seq protocol in addition to 3′-tag counting assay.

Subsequent pooling, cleanup, single primer cDNA PCR amplification,tagmentation and sequencing library prep can be performed in a singletube for 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 91,92, 93, 94, 95, 96, 97, 98, 99, 100 or more than cells. Thus, themethods and compositions described herein are highly amenable tomultiplexing. As an example, set forth in FIG. 5, the methods providedherein enable multiplexing at cellular levels (e.g., 96 samples per96-well plate), Further, the methods enable further multiplexing at theplate level using uniquely barcoded plates (e.g., tagmentation toincorporate a barcode that identifies the 96 well plate). These methodsare amenable to automation and can provide signification cost and timesavings.

It will be appreciated that the order of a sample barcode and UMI on thefirst strand synthesis primer can be varied. For example, in someembodiments, the sample barcode (BC) is positioned 3′ to the UMI. Insome embodiments, the sample barcode (BC) is positioned 5′ to the UMI.In some embodiments, the sample barcode (BC) is directly contiguous withthe UMI. In some embodiments, the sample barcode (BC) is separated fromthe UMI by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 nucleotides. Insome embodiments, the sample barcode (BC) overlaps with the UMI by 1, 2,3, 4, 5, 6, 7, 8, 9, 10 or more than 10 nucleotides.

In some embodiments, tagmentation is performed using a transposasemixture that contains adapter sequences that are not found in the firststrand synthesis primer. Doing so produces tagmentation fragments thatbear different adapter sequences compared to the sequence incorporatedinto the first strand synthesis primer. For example, in someembodiments, the transposase mixture exclusively comprises transposomeshaving one type of adapter sequence. Fragments that were generated bythis type of mixture are referred to herein as “symmetric fragments” anddo not produce sequenceable clusters on an Illumina flowcell. In someembodiments, the transposase mixture may comprise some amount of thesequence incorporated into the first strand synthesis primer, whereinthe amount is low enough to still allow all or substantially all of the3′ cDNA fragments to be amplified and sequenced. For example, theadapter sequences in the transposome mixture may comprise less than0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%,14%, 15%, 16%, 17% 18%, 19%, 20%, or less than 25%, 30%, 35%, 40%, 45%or less than 50% of the sequence incorporated into the first strandsynthesis primer. It will be appreciated that where two transposaseadapters are typically used, either one of the two adapters can beincorporated into the first strand synthesis primer, and the otheradapter can be used during the tagmentation event. For example, in theexemplary embodiments set forth in FIGS. 3A, 3B, 4A and 4B, where oneadapter sequence is incorporated into the first strand synthesis primer,that adapter is not used in the tagmentation mixture. FIGS. 3A and 3Bshow one embodiment where adaptor V2.A14 is incorporated into the firststrand synthesis primer and adapter V2.B15 is used as the transposomeadapter during the tagmentation step. FIGS. 4A and 4B show analternative embodiment where adaptor V2.B15 is incorporated into thefirst strand synthesis primer and adapter V2.A14 is used as thetransposome adapter during the tagmentation step. In both instances, theresulting tagmentation fragments fall in to two categories: symmetricfragments (unable to be amplified and/or sequenced), and asymmetricfragments which can be amplified using a set of V2.A14 and V2.B15primers.

In some embodiments, the number of single cells that can be multiplexedis significantly increased by incorporating indexes into the transposomeadapter sequences. As exemplified in FIG. 5, each individual cell can beidentified using a cell-specific barcode and each set of cells (forexample, a plate of 96 cells) can be contacted with a tagmentationmixture having a plate-specific barcode incorporated into thetransposome adapter sequence. In the example shown in FIG. 5, the cDNAsfrom each of the 96 cells on a plate are pooled prior to tagmentation,and then the tagmented samples are pooled for multiplexed sequencing. Itwill be appreciated that any number of cells can be pooled prior totagmentation, and that use of a 96 well plate is simply one of a varietyof embodiments. For example, a set of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64,66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100,or more than 100, 200, 300, 400, 500, 600, 700, 800, 900 or more than1000, and any intermediate number of cells may be pooled fortagmentation. First strand synthesis may take place in any single ormulti-well vessel, such as a multi-well plate, chip, microfluidicdevice, emulsion, bead mixture, or any other suitable format formultiplex handling of a plurality of cells.

As shown in FIG. 7 (single cells), when gene expression was analyzedusing either symmetric tagmentation (Nextera version V2.B15) compared toasymmetric tagmentation (Nextera version V2.B15/V2.A14) a significantnumber of genes were detected and other metrics were obtained. FIG. 8shows that transcript coverage is almost entirely biased towards the 3′end of the transcripts when performing tagmentation using only onetransposase adaptor (V2.B15) versus tagmentation using two transposaseadaptors (V2.A14 and V2.B15).

In some embodiments, sample and UMI barcoding can be performed bysegregating individual cells into droplets. In some embodiments, thedroplets are segregated from each other in an emulsion. In someembodiments, the droplets are formed and/or manipulated using a dropletactuator. In particular embodiments, one or more droplets comprise adifferent set of barcode-containing first strand synthesis primers. Insome embodiments, each droplet comprise multitude of first strandsynthesis primers, each of these primers have identical sequenceincluding identical barcodes and the barcodes from one droplet differfrom another droplet, while the remaining portion of the first strandsynthesis primer remains the same between the droplets. Thus, in theseembodiments, the barcodes act as identifier for the droplets as well aswell as the single cell encompassed by the droplet. In particularembodiments, one or more droplets comprise a different set ofUMI-containing first strand synthesis primers. Thus, each individualcell that is lysed in each droplet will be identifiable by the barcodesin each droplet. As illustrated in FIG. 11, droplet-based barcoding canbe performed by merging droplets containing single cells with otherdroplets that comprise unique sets of barcodes. This format allowsadditional multiplexing beyond that available in a multiwall format.First strand synthesis and template switching is performed within eachindividual droplet. In some embodiments, two or more droplets can bemerged prior to PCR. Additionally or alternatively, in some embodiments,droplets can be merged prior to tagmentation. Additionally oralternatively, in some embodiments, droplets can be merged after PCR andprior to tagmentation. For example, in some embodiments, after firststrand synthesis is performed in individual droplets, the tagged cDNAscan be merged, thus pooling the cDNAs.

Similarly, in some embodiments, sample and UMI barcoding can beperformed by segregating individual cells with beads that bear a UMIand/or barcode-tagged primer for first strand synthesis. In someembodiments, beads are segregated into droplets in an emulsion. In someembodiments, beads are segregated and manipulated using a dropletactuator. As illustrated in FIG. 12, bead-based barcoding can beperformed by creating a set of beads, each bead bearing a unique set orsets of barcodes.

Whole Transcriptome Sequencing

In some embodiments, the methods provided herein can be utilized toperform whole transcriptome sequencing. In such embodiments, firststrand synthesis is expanded using randomers. As illustrated in FIG. 9A,random priming expands the window of sequenceable fragments to anywherealong the length of the transcript where a randomer can hybridize andprime first strand synthesis. The randomers can include the samecombinations of sample barcodes and unique molecular identifiers inaddition to other adapter and primer binding sites, such as atransposome adapter sequence and/or template switching (TS) primer.Template switching and second strand synthesis can be performed asdescribed above for oligo-dT primed cDNA synthesis. The resulting doublestranded cDNAs can then be subjected to a tagmentation reaction asdescribed above. The difference between use of randomers versus oligo-dTprimers is that a complete, or substantially complete sequence oftranscript can be obtained, rather than the 3′ portion of thetranscript. As shown in FIG. 6 (100 pg RNA), when gene expression wasanalyzed using either oligo-dT compared to mix of oligo-dT and randomersfor first strand synthesis, a significant number of genes were detectedand other metrics were obtained.

One type of byproduct that can occur when using randomers for primingfirst strand synthesis is concatenation byproducts. Specifically, insome situations, as illustrated in FIG. 9B, randomers may hybridize toother random primers, thus outcompeting annealing to RNA transcripts.The cDNA products that result may be further subjected to random primingand a cascade of template switching events can occur. This cascade canlead to formation of a byproduct of concatemers that may be dependent onthe presence of the template switching oligonucleotide.

In order to reduce and/or minimize the formation of such byproducts, avariety of primer designs are presented herein which reduce thelikelihood of byproduct formation. Exemplary designs are set forth inFIG. 10, although it will be appreciated that the scope of the primercompositions set forth herein extends beyond the examples set forth inthe figure. In one embodiment, primers can be configured to form ahairpin to prevent or minimize randomer priming to any portion of thebarcode, adapter or amplification primer binding regions. Adouble-stranded portion formed in the primer itself can thus out-competerandomer hybridization. In some embodiments, the double-stranded portionof the hairpin comprises the mosaic end (ME) sequence of the transposaseadapter. Alternatively or additionally, in some embodiments, the doublestranded portion can comprise some or all of the transposase adaptersequence, beyond the mosaic end sequence. In some embodiments, theadapter sequence can be completely replaced with a short RNA sequence,thus reducing the length of the primer and minimizing the potential fora randomer to hybridize to the primer. In some embodiments, a complementof the short RNA sequence is provided at or near the 5′ end of theprimer, thus enabling formation of a hairpin. In some embodiments, adouble-stranded region is formed by annealing a complementaryoligonucleotide to the adapter and/or primer portion, thus preventing orminimizing randomer priming to any portion of the barcode, adapter oramplification primer binding regions and thereby reducing or avoidingconcatenation byproducts.

Randomer or Random Primer

As used herein, the terms “randomer” and “random primer: are usedinterchangeably. The term randomer refers to The random primers that canexhibit fourfold degeneracy at each position.

In some embodiments, the randomers comprises nucleic acid primers thatare any of a variety of random sequence lengths, as known in the art.For example, the randomers can comprise a random sequence that is 3, 4,5, 6, 7, 8, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or morenucleotides long. In certain embodiments, the plurality of randomprimers can comprise randomers of various lengths. In certainembodiments, the plurality of randomers can comprise randomers that areof equal length. In certain embodiments, the plurality of randomers cancomprise a random sequence that is about 5 to about 18 nucleotides long.In some embodiments, the plurality of randomers comprises randomhexamers. Random primers, and particularly random hexamers, arecommercially available and widely used in amplification reactions suchas Multiple Displacement Amplification (MDA), as exemplified by REPLI-gwhole genome amplification kits (QIAGEN, Valencia, Calif.). It will beappreciated that any suitable length of randomers may be used in themethods and compositions presented herein.

Exemplary randomer sequences comprising a 3′-random sequence portion anda 5′-defined sequence portion are shown below:

(SEQ ID NO: 6) GTGTAGATCT CGGTGGTCGC CGTATCATTN NNNN (SEQ ID NO: 4)GTGTAGATCT CGGTGGTCGC CGTATCATTN NNNNN (SEQ ID NO: 7GTGTAGATCT CGGTGGTCGC CGTATCATTN NNNNNN (SEQ ID NO: 8)GTGTAGATCT CGGTGGTCGC CGTATCATTN NNNNNNN (SEQ ID NO: 9)GTGTAGATCT CGGTGGTCGC CGTATCATTN NNNNNNNN (SEQ ID NO: 10)ATCTCGTATG CCGTCTTCTG CTTGNNNNN (SEQ ID NO: 5)ATCTCGTATG CCGTCTTCTG CTTGNNNNNN (SEQ ID NO: 11)ATCTCGTATG CCGTCTTCTG CTTGNNNNNNN (SEQ ID NO: 12)ATCTCGTATG CCGTCTTCTG CTTGNNNNNNNN (SEQ ID NO: 13)ATCTCGTATG CCGTCTTCTG CTTGNNNNNNNNN

As used herein, the term “SMART-Seq Plus” means a method of preparingcDNA from RNA using a first strand synthesis primer comprising a firstamplification primer binding site, a randomer comprising a firstamplification primer binding site, and an oligonucleotide switchingoligonucleotide that is partially complimentary to the first strand ofthe cDNA and comprises a second amplification primer binding site. Insome embodiments, the first strand synthesis primer comprises anoligo(dT) portion.

Barcodes and UMIs

As used herein, the term “barcode” or “BC” refers to a nucleic acid tagthat can be used to identify a sample or source of the nucleic acidmaterial. Thus, where nucleic acid samples are derived from multiplesources, the nucleic acids in each nucleic acid sample can be taggedwith different nucleic acid tags such that the source of the sample canbe identified. Barcodes, also commonly referred to indexes, tags, andthe like, are well known to those of skill in the art. Any suitablebarcode or set of barcodes can be used, as known in the art and asexemplified by the disclosures of U.S. Pat. No. 8,053,192 and PCT Pub.WO05/068656, which are incorporated herein by reference in theirentireties. Barcoding of single cells can be performed as described, forexample in the disclosure of U.S. 2013/0274117, which is incorporatedherein by reference in its entirety.

Nucleic acids from more than one source can incorporate a variable tagsequence. This tag sequence can be up to 100 nucleotides in length (basepairs if referring to double stranded molecules), preferably 1 to 10nucleotides in length, most preferably 4, 5 or 6 nucleotides in lengthand comprises combinations of nucleotides. For example, in oneembodiment, if six base-pairs are chosen to form the tag and apermutation of four different nucleotides used, then a total of 4096nucleic acid anchors (e.g. hairpins), each with a unique 6 base tag canbe made.

As used herein, the terms UMI, unique identifier, and unique molecularidentifier refer to a unique nucleic acid sequence that is attached toeach of a plurality of nucleic acid molecules. When incorporated into anucleic acid molecule, for example during first strand cDNA synthesis, aUMI can be used to correct for subsequent amplification bias by directlycounting unique molecular identifiers (UMIs) that are sequenced afteramplification. The design, incorporation and application of UMIs cantake place as known in the art, as exemplified by, for example, thedisclosures of WO 2012/142213, Islam et al. Nat. Methods (2014)11:163-166, and Kivioja, T. et al. Nat. Methods (2012) 9: 72-74, each ofwhich is incorporated by reference in its entirety.

Tagmentation

As used herein, the term “tagmentation” refers to the modification ofDNA by a transposome complex comprising transposase enzyme complexedwith adaptors comprising transposon end sequence. Tagmentation resultsin the simultaneous fragmentation of the DNA and ligation of theadaptors to the 5′ ends of both strands of duplex fragments. Following apurification step to remove the transposase enzyme, additional sequencescan be added to the ends of the adapted fragments, for example by PCR,ligation, or any other suitable methodology known to those of skill inthe art.

The method of the invention can use any transposase that can accept atransposase end sequence and fragment a target nucleic acid, attaching atransferred end, but not a non-transferred end. A “transposome” iscomprised of at least a transposase enzyme and a transposase recognitionsite. In some such systems, termed “transposomes”, the transposase canform a functional complex with a transposon recognition site that iscapable of catalyzing a transposition reaction. The transposase orintegrase may bind to the transposase recognition site and insert thetransposase recognition site into a target nucleic acid in a processsometimes termed “tagmentation”. In some such insertion events, onestrand of the transposase recognition site may be transferred into thetarget nucleic acid.

In standard sample preparation methods, each template contains anadaptor at either end of the insert and often a number of steps arerequired to both modify the DNA or RNA and to purify the desiredproducts of the modification reactions. These steps are performed insolution prior to the addition of the adapted fragments to a flowcellwhere they are coupled to the surface by a primer extension reactionthat copies the hybridized fragment onto the end of a primer covalentlyattached to the surface. These ‘seeding’ templates then give rise tomonoclonal clusters of copied templates through several cycles ofamplification.

The number of steps required to transform DNA into adaptor-modifiedtemplates in solution ready for cluster formation and sequencing can beminimized by the use of transposase mediated fragmentation and tagging.

In some embodiments, transposon based technology can be utilized forfragmenting DNA, for example as exemplified in the workflow for Nextera™DNA sample preparation kits (Illumina, Inc.) wherein genomic DNA can befragmented by an engineered transposome that simultaneously fragmentsand tags input DNA (“tagmentation”) thereby creating a population offragmented nucleic acid molecules which comprise unique adaptersequences at the ends of the fragments.

Some embodiments can include the use of a hyperactive Tn5 transposaseand a Tn5-type transposase recognition site (Goryshin and Reznikoff, J.Biol. Chem., 273:7367 (1998)), or MuA transposase and a Mu transposaserecognition site comprising R1 and R2 end sequences (Mizuuchi, K., Cell,35: 785, 1983; Savilahti, H, et al., EMBO J., 14: 4893, 1995). Anexemplary transposase recognition site that forms a complex with ahyperactive Tn5 transposase (e.g., EZ-Tn5™ Transposase, EpicentreBiotechnologies, Madison, Wis.).

More examples of transposition systems that can be used with certainembodiments provided herein include Staphylococcus aureus Tn552 (Colegioet al., J. Bacteriol., 183: 2384-8, 2001; Kirby C et al., Mol.Microbiol., 43: 173-86, 2002), Ty1 (Devine & Boeke, Nucleic Acids Res.,22: 3765-72, 1994 and International Publication WO 95/23875), TransposonTn7 (Craig, N L, Science. 271: 1512, 1996; Craig, N L, Review in: CurrTop Microbiol Immunol., 204:27-48, 1996), Tn/O and IS10 (Kleckner N, etal., Curr Top Microbiol Immunol., 204:49-82, 1996), Mariner transposase(Lampe D J, et al., EMBO J., 15: 5470-9, 1996), Tc1 (Plasterk R H, Curr.Topics Microbiol. Immunol., 204: 125-43, 1996), P Element (Gloor, G B,Methods Mol. Biol., 260: 97-114, 2004), Tn3 (Ichikawa & Ohtsubo, J Biol.Chem. 265:18829-32, 1990), bacterial insertion sequences (Ohtsubo &Sekine, Curr. Top. Microbiol. Immunol. 204: 1-26, 1996), retroviruses(Brown, et al., Proc Natl Acad Sci USA, 86:2525-9, 1989), andretrotransposon of yeast (Boeke & Corces, Annu Rev Microbiol. 43:403-34,1989). More examples include IS5, Tn10, Tn903, IS911, and engineeredversions of transposase family enzymes (Zhang et al., (2009) PLoS Genet.5:e1000689. Epub 2009 Oct. 16; Wilson C. et al (2007) J. Microbiol.Methods 71:332-5).

Briefly, a “transposition reaction” is a reaction wherein one or moretransposons are inserted into target nucleic acids at random sites oralmost random sites. Essential components in a transposition reactionare a transposase and DNA oligonucleotides that exhibit the nucleotidesequences of a transposon, including the transferred transposon sequenceand its complement (i.e., the non-transferred transposon end sequence)as well as other components needed to form a functional transposition ortransposome complex. The DNA oligonucleotides can further compriseadditional sequences (e.g., adaptor or primer sequences) as needed ordesired. Briefly, in vitro transposition can be initiated by contactinga transposome complex and a target DNA. Exemplary transpositionprocedures and systems that can be readily adapted for use with thetransposases of the present disclosure are described, for example, in WO10/048605; US 2012/0301925; US 2013/0143774, each of which isincorporated herein by reference in its entirety.

The adapters that are added to the 5′ and/or 3′ end of a nucleic acidcan comprise a universal sequence. A universal sequence is a region ofnucleotide sequence that is common to, i.e., shared by, two or morenucleic acid molecules. Optionally, the two or more nucleic acidmolecules also have regions of sequence differences. Thus, for example,the 5′ adapters can comprise identical or universal nucleic acidsequences and the 3′ adapters can comprise identical or universalsequences. A universal sequence that may be present in different membersof a plurality of nucleic acid molecules can allow the replication oramplification of multiple different sequences using a single universalprimer that is complementary to the universal sequence. Some universalprimer sequences used in examples presented herein include the V2.A14and V2.B15 Nextera™ sequences. However, it will be readily appreciatedthat any suitable adapter sequence can be utilized in the methods andcompositions presented herein. For example, Tn5 Mosaic End Sequence A14(Tn5MEA) and/or Tn5 Mosaic End Sequence B15 (Tn5MEB), including thecomplementary non transferred sequence (NTS) as set forth below, can beused in the methods provided herein.

Tn5MEA: (SEQ ID NO: 1) 5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3′ Tn5MEB:(SEQ ID NO: 2) 5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3′ Tn5 NTS:(SEQ ID NO: 3) 5′-CTGTCTCTTATACACATCT-3′Barcodes and UMIs in Droplets

In some embodiments, primers bearing sample barcodes can be in solution.Additionally or alternatively, primers bearing UMI sequences can be insolution. For example, the solid support can be one or more droplets.Thus, in certain embodiments, a plurality of droplets can be presented,wherein each droplet in the plurality bears a unique sample barcodeand/or UMI sequences, each of which are unique to a molecule. Thus, aperson of ordinary skill in the art will understand that in someembodiments, the barcodes are unique to a droplet and the UMI are uniqueto a molecule such that the UMI are repeated many times within acollection of droplets. In some embodiments, individual cells arecontacted with a droplet having a unique set of sample barcodes and/orUMI sequences in order to identify the individual cell. In someembodiments, lysates from individual cells are contacted with a droplethaving a unique set of sample barcodes and/or UMI sequences in order toidentify the individual cell lysates. In some embodiments, purifiednucleic acid from individual cells are contacted with a droplet having aunique set of sample barcodes and/or UMI sequences in order to identifythe purified nucleic acid from the individual cell.

Any suitable system for forming and manipulating droplets can be used inthe embodiments presented herein where each droplet in a plurality ofdroplets bears a unique set of sample barcodes and/or UMI sequences. Forexample, a droplet actuator may be used.

“Droplet Actuator” means a device for manipulating droplets. Forexamples of droplet actuators, see Pamula et al., U.S. Pat. No.6,911,132, entitled “Apparatus for Manipulating Droplets byElectrowetting-Based Techniques,” issued on Jun. 28, 2005; Pamula etal., U.S. Patent Pub. No. 20060194331, entitled “Apparatuses and Methodsfor Manipulating Droplets on a Printed Circuit Board,” published on Aug.31, 2006; Pollack et al., International Patent Pub. No. WO/2007/120241,entitled “Droplet-Based Biochemistry,” published on Oct. 25, 2007;Shenderov, U.S. Pat. No. 6,773,566, entitled “Electrostatic Actuatorsfor Microfluidics and Methods for Using Same,” issued on Aug. 10, 2004;Shenderov, U.S. Pat. No. 6,565,727, entitled “Actuators forMicrofluidics Without Moving Parts,” issued on May 20, 2003; Kim et al.,U.S. Patent Pub. No. 20030205632, entitled “Electrowetting-drivenMicropumping,” published on Nov. 6, 2003; Kim et al., U.S. Patent Pub.No. 20060164490, entitled “Method and Apparatus for Promoting theComplete Transfer of Liquid Drops from a Nozzle,” published on Jul. 27,2006; Kim et al., U.S. Patent Pub. No. 20070023292, entitled “SmallObject Moving on Printed Circuit Board,” published on Feb. 1, 2007; Shahet al., U.S. Patent Pub. No. 20090283407, entitled “Method for UsingMagnetic Particles in Droplet Microfluidics,” published on Nov. 19,2009; Kim et al., U.S. Patent Pub. No. 20100096266, entitled “Method andApparatus for Real-time Feedback Control of Electrical Manipulation ofDroplets on Chip,” published on Apr. 22, 2010; Velev, U.S. Pat. No.7,547,380, entitled “Droplet Transportation Devices and Methods Having aFluid Surface,” issued on Jun. 16, 2009; Sterling et al., U.S. Pat. No.7,163,612, entitled “Method, Apparatus and Article for MicrofluidicControl via Electrowetting, for Chemical, Biochemical and BiologicalAssays and the Like,” issued on Jan. 16, 2007; Becker et al., U.S. Pat.No. 7,641,779, entitled “Method and Apparatus for Programmable FluidicProcessing,” issued on Jan. 5, 2010; Becker et al., U.S. Pat. No.6,977,033, entitled “Method and Apparatus for Programmable FluidicProcessing,” issued on Dec. 20, 2005; Decre et al., U.S. Pat. No.7,328,979, entitled “System for Manipulation of a Body of Fluid,” issuedon Feb. 12, 2008; Yamakawa et al., U.S. Patent Pub. No. 20060039823,entitled “Chemical Analysis Apparatus,” published on Feb. 23, 2006; Wu,U.S. Patent Pub. No. 20110048951, entitled “Digital Microfluidics BasedApparatus for Heat-exchanging Chemical Processes,” published on Mar. 3,2011; Fouillet et al., U.S. Patent Pub. No. 20090192044, entitled“Electrode Addressing Method,” published on Jul. 30, 2009; Fouillet etal., U.S. Pat. No. 7,052,244, entitled “Device for Displacement of SmallLiquid Volumes Along a Micro-catenary Line by Electrostatic Forces,”issued on May 30, 2006; Marchand et al., U.S. Patent Pub. No.20080124252, entitled “Droplet Microreactor,” published on May 29, 2008;Adachi et al., U.S. Patent Pub. No. 20090321262, entitled “LiquidTransfer Device,” published on Dec. 31, 2009; Roux et al., U.S. PatentPub. No. 20050179746, entitled “Device for Controlling the Displacementof a Drop Between Two or Several Solid Substrates,” published on Aug.18, 2005; and Dhindsa et al., “Virtual Electrowetting Channels:Electronic Liquid Transport with Continuous Channel Functionality,” LabChip, 10:832-836 (2010), the entire disclosures of which areincorporated herein by reference. Certain droplet actuators will includeone or more substrates arranged with a droplet operations gaptherebetween and electrodes associated with (e.g., layered on, attachedto, and/or embedded in) the one or more substrates and arranged toconduct one or more droplet operations. For example, certain dropletactuators will include a base (or bottom) substrate, droplet operationselectrodes associated with the substrate, one or more dielectric layersatop the substrate and/or electrodes, and optionally one or morehydrophobic layers atop the substrate, dielectric layers and/or theelectrodes forming a droplet operations surface. A top substrate mayalso be provided, which is separated from the droplet operations surfaceby a gap, commonly referred to as a droplet operations gap. Variouselectrode arrangements on the top and/or bottom substrates are discussedin the above-referenced patents and applications and certain novelelectrode arrangements are discussed in the description of the presentdisclosure. During droplet operations it is preferred that dropletsremain in continuous contact or frequent contact with a ground orreference electrode. A ground or reference electrode may be associatedwith the top substrate facing the gap, the bottom substrate facing thegap, in the gap. Where electrodes are provided on both substrates,electrical contacts for coupling the electrodes to a droplet actuatorinstrument for controlling or monitoring the electrodes may beassociated with one or both plates. In some cases, electrodes on onesubstrate are electrically coupled to the other substrate so that onlyone substrate is in contact with the droplet actuator. In oneembodiment, a conductive material (e.g., an epoxy, such as MASTER BOND™Polymer System EP79, available from Master Bond, Inc., Hackensack, N.J.)provides the electrical connection between electrodes on one substrateand electrical paths on the other substrates, e.g., a ground electrodeon a top substrate may be coupled to an electrical path on a bottomsubstrate by such a conductive material. Where multiple substrates areused, a spacer may be provided between the substrates to determine theheight of the gap therebetween and define on-actuator dispensingreservoirs. The spacer height may, for example, be at least about 5 μm,100 μm, 200 μm, 250 μm, 275 μm or more. Alternatively or additionallythe spacer height may be at most about 600 μm, 400 μm, 350 μm, 300 μm,or less. The spacer may, for example, be formed of a layer ofprojections form the top or bottom substrates, and/or a materialinserted between the top and bottom substrates. One or more openings maybe provided in the one or more substrates for forming a fluid paththrough which liquid may be delivered into the droplet operations gap.The one or more openings may in some cases be aligned for interactionwith one or more electrodes, e.g., aligned such that liquid flowedthrough the opening will come into sufficient proximity with one or moredroplet operations electrodes to permit a droplet operation to beeffected by the droplet operations electrodes using the liquid. The base(or bottom) and top substrates may in some cases be formed as oneintegral component. One or more reference electrodes may be provided onthe base (or bottom) and/or top substrates and/or in the gap. Examplesof reference electrode arrangements are provided in the above referencedpatents and patent applications. In various embodiments, themanipulation of droplets by a droplet actuator may be electrodemediated, e.g., electrowetting mediated or dielectrophoresis mediated orCoulombic force mediated. Examples of other techniques for controllingdroplet operations that may be used in the droplet actuators of thepresent disclosure include using devices that induce hydrodynamicfluidic pressure, such as those that operate on the basis of mechanicalprinciples (e.g. external syringe pumps, pneumatic membrane pumps,vibrating membrane pumps, vacuum devices, centrifugal forces,piezoelectric/ultrasonic pumps and acoustic forces); electrical ormagnetic principles (e.g. electroosmotic flow, electrokinetic pumps,ferrofluidic plugs, electrohydrodynamic pumps, attraction or repulsionusing magnetic forces and magnetohydrodynamic pumps); thermodynamicprinciples (e.g. gas bubble generation/phase-change-induced volumeexpansion); other kinds of surface-wetting principles (e.g.electrowetting, and optoelectrowetting, as well as chemically,thermally, structurally and radioactively induced surface-tensiongradients); gravity; surface tension (e.g., capillary action);electrostatic forces (e.g., electroosmotic flow); centrifugal flow(substrate disposed on a compact disc and rotated); magnetic forces(e.g., oscillating ions causes flow); magnetohydrodynamic forces; andvacuum or pressure differential. In certain embodiments, combinations oftwo or more of the foregoing techniques may be employed to conduct adroplet operation in a droplet actuator of the present disclosure.Similarly, one or more of the foregoing may be used to deliver liquidinto a droplet operations gap, e.g., from a reservoir in another deviceor from an external reservoir of the droplet actuator (e.g., a reservoirassociated with a droplet actuator substrate and a flow path from thereservoir into the droplet operations gap). Droplet operations surfacesof certain droplet actuators of the present disclosure may be made fromhydrophobic materials or may be coated or treated to make themhydrophobic. For example, in some cases some portion or all of thedroplet operations surfaces may be derivatized with low surface-energymaterials or chemistries, e.g., by deposition or using in situ synthesisusing compounds such as poly- or per-fluorinated compounds in solutionor polymerizable monomers. Examples include TEFLON® AF (available fromDuPont, Wilmington, Del.), members of the cytop family of materials,coatings in the FLUOROPEL® family of hydrophobic and superhydrophobiccoatings (available from Cytonix Corporation, Beltsville, Md.), silanecoatings, fluorosilane coatings, hydrophobic phosphonate derivatives(e.g., those sold by Aculon, Inc), and NOVEC™ electronic coatings(available from 3M Company, St. Paul, Minn.), other fluorinated monomersfor plasma-enhanced chemical vapor deposition (PECVD), andorganosiloxane (e.g., SiOC) for PECVD. In some cases, the dropletoperations surface may include a hydrophobic coating having a thicknessranging from about 10 nm to about 1,000 nm. Moreover, in someembodiments, the top substrate of the droplet actuator includes anelectrically conducting organic polymer, which is then coated with ahydrophobic coating or otherwise treated to make the droplet operationssurface hydrophobic. For example, the electrically conducting organicpolymer that is deposited onto a plastic substrate may bepoly(3,4-ethylenedioxythiophene)poly(styrenesulfonate) (PEDOT:PSS).Other examples of electrically conducting organic polymers andalternative conductive layers are described in Pollack et al.,International Patent Pub. No. WO/2011/002957, entitled “Droplet ActuatorDevices and Methods,” published on Jan. 6, 2011, the entire disclosureof which is incorporated herein by reference. One or both substrates maybe fabricated using a printed circuit board (PCB), glass, indium tinoxide (ITO)-coated glass, and/or semiconductor materials as thesubstrate. When the substrate is ITO-coated glass, the ITO coating ispreferably a thickness of at least about 20 nm, 50 nm, 75 nm, 100 nm ormore. Alternatively or additionally the thickness can be at most about200 nm, 150 nm, 125 nm or less. In some cases, the top and/or bottomsubstrate includes a PCB substrate that is coated with a dielectric,such as a polyimide dielectric, which may in some cases also be coatedor otherwise treated to make the droplet operations surface hydrophobic.When the substrate includes a PCB, the following materials are examplesof suitable materials: MITSUI™ BN-300 (available from MITSUI ChemicalsAmerica, Inc., San Jose Calif.); ARLON™ 11N (available from Arlon, Inc,Santa Ana, Calif.).; NELCO® N4000-6 and N5000-30/32 (available from ParkElectrochemical Corp., Melville, N.Y.); ISOLA™ FR406 (available fromIsola Group, Chandler, Ariz.), especially IS620; fluoropolymer family(suitable for fluorescence detection since it has low backgroundfluorescence); polyimide family; polyester; polyethylene naphthalate;polycarbonate; polyetheretherketone; liquid crystal polymer;cyclo-olefin copolymer (COC); cyclo-olefin polymer (COP); aramid;THERMOUNT® nonwoven aramid reinforcement (available from DuPont,Wilmington, Del.); NOMEX® brand fiber (available from DuPont,Wilmington, Del.); and paper. Various materials are also suitable foruse as the dielectric component of the substrate. Examples include:vapor deposited dielectric, such as PARYLENE™ C (especially on glass),PARYLENE™ N, and PARYLENE™ HT (for high temperature, ˜300° C.)(available from Parylene Coating Services, Inc., Katy, Tex.); TEFLON® AFcoatings; cytop; soldermasks, such as liquid photoimageable soldermasks(e.g., on PCB) like TAIYO™ PSR4000 series, TAIYO™ PSR and AUS series(available from Taiyo America, Inc. Carson City, Nev.) (good thermalcharacteristics for applications involving thermal control), andPROBIMER™ 8165 (good thermal characteristics for applications involvingthermal control (available from Huntsman Advanced Materials AmericasInc., Los Angeles, Calif.); dry film soldermask, such as those in theVACREL® dry film soldermask line (available from DuPont, Wilmington,Del.); film dielectrics, such as polyimide film (e.g., KAPTON® polyimidefilm, available from DuPont, Wilmington, Del.), polyethylene, andfluoropolymers (e.g., FEP), polytetrafluoroethylene; polyester;polyethylene naphthalate; cyclo-olefin copolymer (COC); cyclo-olefinpolymer (COP); any other PCB substrate material listed above; blackmatrix resin; polypropylene; and black flexible circuit materials, suchas DuPont™ Pyralux® HXC and DuPont™ Kapton® MBC (available from DuPont,Wilmington, Del.). Droplet transport voltage and frequency may beselected for performance with reagents used in specific assay protocols.Design parameters may be varied, e.g., number and placement ofon-actuator reservoirs, number of independent electrode connections,size (volume) of different reservoirs, placement of magnets/bead washingzones, electrode size, inter-electrode pitch, and gap height (betweentop and bottom substrates) may be varied for use with specific reagents,protocols, droplet volumes, etc. In some cases, a substrate of thepresent disclosure may be derivatized with low surface-energy materialsor chemistries, e.g., using deposition or in situ synthesis using poly-or per-fluorinated compounds in solution or polymerizable monomers.Examples include TEFLON® AF coatings and FLUOROPEL® coatings for dip orspray coating, other fluorinated monomers for plasma-enhanced chemicalvapor deposition (PECVD), and organosiloxane (e.g., SiOC) for PECVD.Additionally, in some cases, some portion or the entire dropletoperations surface may be coated with a substance for reducingbackground noise, such as background fluorescence from a PCB substrate.For example, the noise-reducing coating may include a black matrixresin, such as the black matrix resins available from Toray industries,Inc., Japan. Electrodes of a droplet actuator are typically controlledby a controller or a processor, which is itself provided as part of asystem, which may include processing functions as well as data andsoftware storage and input and output capabilities. Reagents may beprovided on the droplet actuator in the droplet operations gap or in areservoir fluidly coupled to the droplet operations gap. The reagentsmay be in liquid form, e.g., droplets, or they may be provided in areconstitutable form in the droplet operations gap or in a reservoirfluidly coupled to the droplet operations gap. Reconstitutable reagentsmay typically be combined with liquids for reconstitution. An example ofreconstitutable reagents suitable for use with the methods and apparatusset forth herein includes those described in Meathrel et al., U.S. Pat.No. 7,727,466, entitled “Disintegratable Films for Diagnostic Devices,”issued on Jun. 1, 2010, the entire disclosure of which is incorporatedherein by reference.

“Activate,” with reference to one or more electrodes, means affecting achange in the electrical state of the one or more electrodes which, inthe presence of a droplet, results in a droplet operation. Activation ofan electrode can be accomplished using alternating current (AC) ordirect current (DC). Any suitable voltage may be used. For example, anelectrode may be activated using a voltage which is greater than about150 V, or greater than about 200 V, or greater than about 250 V, or fromabout 275 V to about 1000 V, or about 300 V. Where an AC signal is used,any suitable frequency may be employed. For example, an electrode may beactivated using an AC signal having a frequency from about 1 Hz to about10 MHz, or from about 10 Hz to about 60 Hz, or from about 20 Hz to about40 Hz, or about 30 Hz.

“Bead,” with respect to beads on a droplet actuator, means any bead orparticle that is capable of interacting with a droplet on or inproximity with a droplet actuator. Beads may be any of a wide variety ofshapes, such as spherical, generally spherical, egg shaped, disc shaped,cubical, amorphous and other three dimensional shapes. The bead may, forexample, be capable of being subjected to a droplet operation in adroplet on a droplet actuator or otherwise configured with respect to adroplet actuator in a manner which permits a droplet on the dropletactuator to be brought into contact with the bead on the dropletactuator and/or off the droplet actuator. Beads may be provided in adroplet, in a droplet operations gap, or on a droplet operationssurface. Beads may be provided in a reservoir that is external to adroplet operations gap or situated apart from a droplet operationssurface, and the reservoir may be associated with a flow path thatpermits a droplet including the beads to be brought into a dropletoperations gap or into contact with a droplet operations surface. Beadsmay be manufactured using a wide variety of materials, including forexample, resins, and polymers. The beads may be any suitable size,including for example, microbeads, microparticles, nanobeads andnanoparticles. In some cases, beads are magnetically responsive; inother cases beads are not significantly magnetically responsive. Formagnetically responsive beads, the magnetically responsive material mayconstitute substantially all of a bead, a portion of a bead, or only onecomponent of a bead. The remainder of the bead may include, among otherthings, polymeric material, coatings, and moieties which permitattachment of an assay reagent. Examples of suitable beads include flowcytometry microbeads, polystyrene microparticles and nanoparticles,functionalized polystyrene microparticles and nanoparticles, coatedpolystyrene microparticles and nanoparticles, silica microbeads,fluorescent microspheres and nanospheres, functionalized fluorescentmicrospheres and nanospheres, coated fluorescent microspheres andnanospheres, color dyed microparticles and nanoparticles, magneticmicroparticles and nanoparticles, superparamagnetic microparticles andnanoparticles (e.g., DYNABEADS® particles, available from InvitrogenGroup, Carlsbad, Calif.), fluorescent microparticles and nanoparticles,coated magnetic microparticles and nanoparticles, ferromagneticmicroparticles and nanoparticles, coated ferromagnetic microparticlesand nanoparticles, and those described in Watkins et al., U.S. PatentPub. No. 20050260686, entitled “Multiplex Flow Assays Preferably withMagnetic Particles as Solid Phase,” published on Nov. 24, 2005;Chandler., U.S. Patent Pub. No. 20030132538, entitled “Encapsulation ofDiscrete Quanta of Fluorescent Particles,” published on Jul. 17, 2003;Chandler et al., U.S. Patent Pub. No. 20050118574, entitled “MultiplexedAnalysis of Clinical Specimens Apparatus and Method,” published on Jun.2, 2005; Chandler et al., U.S. Patent Pub. No. 20050277197, entitled“Microparticles with Multiple Fluorescent Signals and Methods of UsingSame,” published on Dec. 15, 2005; and Chandler et al., U.S. Patent Pub.No. 20060159962, entitled “Magnetic Microspheres for use inFluorescence-based Applications,” published on Jul. 20, 2006, the entiredisclosures of which are incorporated herein by reference for theirteaching concerning beads and magnetically responsive materials andbeads. Beads may be pre-coupled with a biomolecule or other substancethat is able to bind to and form a complex with a biomolecule. Beads maybe pre-coupled with an antibody, protein or antigen, DNA/RNA probe orany other molecule with an affinity for a desired target. Examples ofdroplet actuator techniques for immobilizing magnetically responsivebeads and/or non-magnetically responsive beads and/or conducting dropletoperations protocols using beads are described in Pollack et al., U.S.Patent Pub. No. 20080053205, entitled “Droplet-Based Particle Sorting,”published on Mar. 6, 2008; U.S. Patent App. No. 61/039,183, entitled“Multiplexing Bead Detection in a Single Droplet,” filed on Mar. 25,2008; Pamula et al., U.S. Patent App. No. 61/047,789, entitled “DropletActuator Devices and Droplet Operations Using Beads,” filed on Apr. 25,2008; U.S. Patent App. No. 61/086,183, entitled “Droplet ActuatorDevices and Methods for Manipulating Beads,” filed on Aug. 5, 2008;Eckhardt et al., International Patent Pub. No. WO/2008/098236, entitled“Droplet Actuator Devices and Methods Employing Magnetic Beads,”published on Aug. 14, 2008; Grichko et al., International Patent Pub.No. WO/2008/134153, entitled “Bead-based Multiplexed Analytical Methodsand Instrumentation,” published on Nov. 6, 2008; Eckhardt et al.,International Patent Pub. No. WO/2008/116221, “Bead Sorting on a DropletActuator,” published on Sep. 25, 2008; and Eckhardt et al.,International Patent Pub. No. WO/2007/120241, entitled “Droplet-basedBiochemistry,” published on Oct. 25, 2007, the entire disclosures ofwhich are incorporated herein by reference. Bead characteristics may beemployed in the multiplexing aspects of the present disclosure. Examplesof beads having characteristics suitable for multiplexing, as well asmethods of detecting and analyzing signals emitted from such beads, maybe found in Whitman et al., U.S. Patent Pub. No. 20080305481, entitled“Systems and Methods for Multiplex Analysis of PCR in Real Time,”published on Dec. 11, 2008; Roth, U.S. Patent Pub. No. 20080151240,“Methods and Systems for Dynamic Range Expansion,” published on Jun. 26,2008; Sorensen et al., U.S. Patent Pub. No. 20070207513, entitled“Methods, Products, and Kits for Identifying an Analyte in a Sample,”published on Sep. 6, 2007; Roth, U.S. Patent Pub. No. 20070064990,entitled “Methods and Systems for Image Data Processing,” published onMar. 22, 2007; Chandler et al., U.S. Patent Pub. No. 20060159962,entitled “Magnetic Microspheres for use in Fluorescence-basedApplications,” published on Jul. 20, 2006; Chandler et al., U.S. PatentPub. No. 20050277197, entitled “Microparticles with Multiple FluorescentSignals and Methods of Using Same,” published on Dec. 15, 2005; andChandler et al., U.S. Patent Publication No. 20050118574, entitled“Multiplexed Analysis of Clinical Specimens Apparatus and Method,”published on Jun. 2, 2005, the entire disclosures of which areincorporated herein by reference.

“Droplet” means a volume of liquid on a droplet actuator. Typically, adroplet is at least partially bounded by a filler fluid. For example, adroplet may be completely surrounded by a filler fluid or may be boundedby filler fluid and one or more surfaces of the droplet actuator. Asanother example, a droplet may be bounded by filler fluid, one or moresurfaces of the droplet actuator, and/or the atmosphere. As yet anotherexample, a droplet may be bounded by filler fluid and the atmosphere.Droplets may, for example, be aqueous or non-aqueous or may be mixturesor emulsions including aqueous and non-aqueous components. Droplets maytake a wide variety of shapes; nonlimiting examples include generallydisc shaped, slug shaped, truncated sphere, ellipsoid, spherical,partially compressed sphere, hemispherical, ovoid, cylindrical,combinations of such shapes, and various shapes formed during dropletoperations, such as merging or splitting or formed as a result ofcontact of such shapes with one or more surfaces of a droplet actuator.For examples of droplet fluids that may be subjected to dropletoperations using the approach of the present disclosure, see Eckhardt etal., International Patent Pub. No. WO/2007/120241, entitled,“Droplet-Based Biochemistry,” published on Oct. 25, 2007, the entiredisclosure of which is incorporated herein by reference.

In various embodiments, a droplet may include a biological sample, suchas whole blood, lymphatic fluid, serum, plasma, sweat, tear, saliva,sputum, cerebrospinal fluid, amniotic fluid, seminal fluid, vaginalexcretion, serous fluid, synovial fluid, pericardial fluid, peritonealfluid, pleural fluid, transudates, exudates, cystic fluid, bile, urine,gastric fluid, intestinal fluid, fecal samples, liquids containingsingle or multiple cells, liquids containing organelles, fluidizedtissues, fluidized organisms, liquids containing multi-celled organisms,biological swabs and biological washes. Moreover, a droplet may includea reagent, such as water, deionized water, saline solutions, acidicsolutions, basic solutions, detergent solutions and/or buffers. Adroplet can include nucleic acids, such as DNA, genomic DNA, RNA, mRNAor analogs thereof; nucleotides such as deoxyribonucleotides,ribonucleotides or analogs thereof such as analogs having terminatormoieties such as those described in Bentley et al., Nature 456:53-59(2008); Gormley et al., International Patent Pub. No. WO/2013/131962,entitled, “Improved Methods of Nucleic Acid Sequencing,” published onSep. 12, 2013; Barnes et al., U.S. Pat. No. 7,057,026, entitled“Labelled Nucleotides,” issued on Jun. 6, 2006; Kozlov et al.,International Patent Pub. No. WO/2008/042067, entitled, “Compositionsand Methods for Nucleotide Sequencing,” published on Apr. 10, 2008;Rigatti et al., International Patent Pub. No. WO/2013/117595, entitled,“Targeted Enrichment and Amplification of Nucleic Acids on a Support,”published on Aug. 15, 2013; Hardin et al., U.S. Pat. No. 7,329,492,entitled “Methods for Real-Time Single Molecule Sequence Determination,”issued on Feb. 12, 2008; Hardin et al., U.S. Pat. No. 7,211,414,entitled “Enzymatic Nucleic Acid Synthesis: Compositions and Methods forAltering Monomer Incorporation Fidelity,” issued on May 1, 2007; Turneret al., U.S. Pat. No. 7,315,019, entitled “Arrays of OpticalConfinements and Uses Thereof,” issued on Jan. 1, 2008; Xu et al., U.S.Pat. No. 7,405,281, entitled “Fluorescent Nucleotide Analogs and UsesTherefor,” issued on Jul. 29, 2008; and Rank et al., U.S. Patent Pub.No. 20080108082, entitled “Polymerase Enzymes and Reagents for EnhancedNucleic Acid Sequencing,” published on May 8, 2008, the entiredisclosures of which are incorporated herein by reference; enzymes suchas polymerases, ligases, recombinases, or transposases; binding partnerssuch as antibodies, epitopes, streptavidin, avidin, biotin, lectins orcarbohydrates; or other biochemically active molecules. Other examplesof droplet contents include reagents, such as a reagent for abiochemical protocol, such as a nucleic acid amplification protocol, anaffinity-based assay protocol, an enzymatic assay protocol, a sequencingprotocol, and/or a protocol for analyses of biological fluids. A dropletmay include one or more beads.

“Droplet operation” means any manipulation of a droplet on a dropletactuator. A droplet operation may, for example, include: loading adroplet into the droplet actuator; dispensing one or more droplets froma source droplet; splitting, separating or dividing a droplet into twoor more droplets; transporting a droplet from one location to another inany direction; merging or combining two or more droplets into a singledroplet; diluting a droplet; mixing a droplet; agitating a droplet;deforming a droplet; retaining a droplet in position; incubating adroplet; heating a droplet; vaporizing a droplet; cooling a droplet;disposing of a droplet; transporting a droplet out of a dropletactuator; other droplet operations described herein; and/or anycombination of the foregoing. The terms “merge,” “merging,” “combine,”“combining” and the like are used to describe the creation of onedroplet from two or more droplets. It should be understood that whensuch a term is used in reference to two or more droplets, anycombination of droplet operations that are sufficient to result in thecombination of the two or more droplets into one droplet may be used.For example, “merging droplet A with droplet B,” can be achieved bytransporting droplet A into contact with a stationary droplet B,transporting droplet B into contact with a stationary droplet A, ortransporting droplets A and B into contact with each other. The terms“splitting,” “separating” and “dividing” are not intended to imply anyparticular outcome with respect to volume of the resulting droplets(i.e., the volume of the resulting droplets can be the same ordifferent) or number of resulting droplets (the number of resultingdroplets may be 2, 3, 4, 5 or more). The term “mixing” refers to dropletoperations which result in more homogenous distribution of one or morecomponents within a droplet. Examples of “loading” droplet operationsinclude microdialysis loading, pressure assisted loading, roboticloading, passive loading, and pipette loading. Droplet operations may beelectrode-mediated. In some cases, droplet operations are furtherfacilitated by the use of hydrophilic and/or hydrophobic regions onsurfaces and/or by physical obstacles. For examples of dropletoperations, see the patents and patent applications cited above underthe definition of “droplet actuator.” Impedance or capacitance sensingor imaging techniques may sometimes be used to determine or confirm theoutcome of a droplet operation. Examples of such techniques aredescribed in Sturmer et al., U.S. Patent Pub. No. 20100194408, entitled“Capacitance Detection in a Droplet Actuator,” published on Aug. 5,2010, the entire disclosure of which is incorporated herein byreference. Generally speaking, the sensing or imaging techniques may beused to confirm the presence or absence of a droplet at a specificelectrode. For example, the presence of a dispensed droplet at thedestination electrode following a droplet dispensing operation confirmsthat the droplet dispensing operation was effective. Similarly, thepresence of a droplet at a detection spot at an appropriate step in anassay protocol may confirm that a previous set of droplet operations hassuccessfully produced a droplet for detection. Droplet transport timecan be quite fast. For example, in various embodiments, transport of adroplet from one electrode to the next may exceed about 1 sec, or about0.1 sec, or about 0.01 sec, or about 0.001 sec. In one embodiment, theelectrode is operated in AC mode but is switched to DC mode for imaging.It is helpful for conducting droplet operations for the footprint areaof droplet to be similar to electrowetting area; in other words, 1×-,2×-3×-droplets are usefully controlled operated using 1, 2, and 3electrodes, respectively. If the droplet footprint is greater thannumber of electrodes available for conducting a droplet operation at agiven time, the difference between the droplet size and the number ofelectrodes should typically not be greater than 1; in other words, a 2×droplet is usefully controlled using 1 electrode and a 3× droplet isusefully controlled using 2 electrodes. When droplets include beads, itis useful for droplet size to be equal to the number of electrodescontrolling the droplet, e.g., transporting the droplet.

“Filler fluid” means a fluid associated with a droplet operationssubstrate of a droplet actuator, which fluid is sufficiently immisciblewith a droplet phase to render the droplet phase subject toelectrode-mediated droplet operations. For example, the dropletoperations gap of a droplet actuator is typically filled with a fillerfluid. The filler fluid may, for example, be or include low-viscosityoil, such as silicone oil or hexadecane filler fluid. The filler fluidmay be or include a halogenated oil, such as a fluorinated orperfluorinated oil. The filler fluid may fill the entire gap of thedroplet actuator or may coat one or more surfaces of the dropletactuator. Filler fluids may be conductive or non-conductive. Fillerfluids may be selected to improve droplet operations and/or reduce lossof reagent or target substances from droplets, improve formation ofmicrodroplets, reduce cross contamination between droplets, reducecontamination of droplet actuator surfaces, reduce degradation ofdroplet actuator materials, etc. For example, filler fluids may beselected for compatibility with droplet actuator materials. As anexample, fluorinated filler fluids may be usefully employed withfluorinated surface coatings. Fluorinated filler fluids are useful toreduce loss of lipophilic compounds, such as umbelliferone substrateslike 6-hexadecanoylamido-4-methylumbelliferone substrates (e.g., for usein Krabbe, Niemann-Pick, or other assays); other umbelliferonesubstrates are described in Winger et al., U.S. Patent Pub. No.20110118132, entitled “Enzymatic Assays Using Umbelliferone Substrateswith Cyclodextrins in Droplets of Oil,” published on May 19, 2011, theentire disclosure of which is incorporated herein by reference. Examplesof suitable fluorinated oils include those in the Galden line, such asGalden HT170 (bp=170° C., viscosity=1.8 cSt, density=1.77), Galden HT200(bp=200C, viscosity=2.4 cSt, d=1.79), Galden HT230 (bp=230C,viscosity=4.4 cSt, d=1.82) (all from Solvay Solexis); those in the Novecline, such as Novec 7500 (bp=128C, viscosity=0.8 cSt, d=1.61),Fluorinert FC-40 (bp=155° C., viscosity=1.8 cSt, d=1.85), FluorinertFC-43 (bp=174° C., viscosity=2.5 cSt, d=1.86) (both from 3M). Ingeneral, selection of perfluorinated filler fluids is based on kinematicviscosity (<7 cSt is preferred, but not required), and on boiling point(>150° C. is preferred, but not required, for use in DNA/RNA-basedapplications (PCR, etc.)). Filler fluids may, for example, be doped withsurfactants or other additives. For example, additives may be selectedto improve droplet operations and/or reduce loss of reagent or targetsubstances from droplets, formation of microdroplets, crosscontamination between droplets, contamination of droplet actuatorsurfaces, degradation of droplet actuator materials, etc. Composition ofthe filler fluid, including surfactant doping, may be selected forperformance with reagents used in the specific assay protocols andeffective interaction or non-interaction with droplet actuatormaterials. Examples of filler fluids and filler fluid formulationssuitable for use with the methods and apparatus set forth herein areprovided in Srinivasan et al, International Patent Pub. No.WO/2010/027894, entitled “Droplet Actuators, Modified Fluids andMethods,” published on Jun. 3, 2010; Srinivasan et al, InternationalPatent Pub. No. WO/2009/021173, entitled “Use of Additives for EnhancingDroplet Operations,” published on Feb. 12, 2009; Sista et al.,International Patent Pub. No. WO/2008/098236, entitled “Droplet ActuatorDevices and Methods Employing Magnetic Beads,” published on Jan. 15,2009; and Monroe et al., U.S. Patent Pub. No. 20080283414, entitled“Electrowetting Devices,” published on Nov. 20, 2008, the entiredisclosures of which are incorporated herein by reference, as well asthe other patents and patent applications cited herein. Fluorinated oilsmay in some cases be doped with fluorinated surfactants, e.g., ZonylFSO-100 (Sigma-Aldrich) and/or others. A filler fluid is typically aliquid. In some embodiments, a filler gas can be used instead of aliquid.

“Immobilize” with respect to magnetically responsive beads, means thatthe beads are substantially restrained in position in a droplet or infiller fluid on a droplet actuator. For example, in one embodiment,immobilized beads are sufficiently restrained in position in a dropletto permit execution of a droplet splitting operation, yielding onedroplet with substantially all of the beads and one dropletsubstantially lacking in the beads.

“Magnetically responsive” means responsive to a magnetic field.“Magnetically responsive beads” include or are composed of magneticallyresponsive materials. Examples of magnetically responsive materialsinclude paramagnetic materials, ferromagnetic materials, ferrimagneticmaterials, and metamagnetic materials. Examples of suitable paramagneticmaterials include iron, nickel, and cobalt, as well as metal oxides,such as Fe3O4, BaFe12O19, CoO, NiO, Mn2O3, Cr2O3, and CoMnP.

“Reservoir” means an enclosure or partial enclosure configured forholding, storing, or supplying liquid. A droplet actuator system of thepresent disclosure may include on-cartridge reservoirs and/oroff-cartridge reservoirs. On-cartridge reservoirs may be (1) on-actuatorreservoirs, which are reservoirs in the droplet operations gap or on thedroplet operations surface; (2) off-actuator reservoirs, which arereservoirs on the droplet actuator cartridge, but outside the dropletoperations gap, and not in contact with the droplet operations surface;or (3) hybrid reservoirs which have on-actuator regions and off-actuatorregions. An example of an off-actuator reservoir is a reservoir in thetop substrate. An off-actuator reservoir is typically in fluidcommunication with an opening or flow path arranged for flowing liquidfrom the off-actuator reservoir into the droplet operations gap, such asinto an on-actuator reservoir. An off-cartridge reservoir may be areservoir that is not part of the droplet actuator cartridge at all, butwhich flows liquid to some portion of the droplet actuator cartridge.For example, an off-cartridge reservoir may be part of a system ordocking station to which the droplet actuator cartridge is coupledduring operation. Similarly, an off-cartridge reservoir may be a reagentstorage container or syringe which is used to force fluid into anon-cartridge reservoir or into a droplet operations gap. A system usingan off-cartridge reservoir will typically include a fluid passage meanswhereby liquid may be transferred from the off-cartridge reservoir intoan on-cartridge reservoir or into a droplet operations gap.

“Transporting into the magnetic field of a magnet,” “transportingtowards a magnet,” and the like, as used herein to refer to dropletsand/or magnetically responsive beads within droplets, is intended torefer to transporting into a region of a magnetic field capable ofsubstantially attracting magnetically responsive beads in the droplet.Similarly, “transporting away from a magnet or magnetic field,”“transporting out of the magnetic field of a magnet,” and the like, asused herein to refer to droplets and/or magnetically responsive beadswithin droplets, is intended to refer to transporting away from a regionof a magnetic field capable of substantially attracting magneticallyresponsive beads in the droplet, whether or not the droplet ormagnetically responsive beads is completely removed from the magneticfield. It will be appreciated that in any of such cases describedherein, the droplet may be transported towards or away from the desiredregion of the magnetic field, and/or the desired region of the magneticfield may be moved towards or away from the droplet. Reference to anelectrode, a droplet, or magnetically responsive beads being “within” or“in” a magnetic field, or the like, is intended to describe a situationin which the electrode is situated in a manner which permits theelectrode to transport a droplet into and/or away from a desired regionof a magnetic field, or the droplet or magnetically responsive beadsis/are situated in a desired region of the magnetic field, in each casewhere the magnetic field in the desired region is capable ofsubstantially attracting any magnetically responsive beads in thedroplet. Similarly, reference to an electrode, a droplet, ormagnetically responsive beads being “outside of” or “away from” amagnetic field, and the like, is intended to describe a situation inwhich the electrode is situated in a manner which permits the electrodeto transport a droplet away from a certain region of a magnetic field,or the droplet or magnetically responsive beads is/are situated awayfrom a certain region of the magnetic field, in each case where themagnetic field in such region is not capable of substantially attractingany magnetically responsive beads in the droplet or in which anyremaining attraction does not eliminate the effectiveness of dropletoperations conducted in the region. In various aspects of the presentdisclosure, a system, a droplet actuator, or another component of asystem may include a magnet, such as one or more permanent magnets(e.g., a single cylindrical or bar magnet or an array of such magnets,such as a Halbach array) or an electromagnet or array of electromagnets,to form a magnetic field for interacting with magnetically responsivebeads or other components on chip. Such interactions may, for example,include substantially immobilizing or restraining movement or flow ofmagnetically responsive beads during storage or in a droplet during adroplet operation or pulling magnetically responsive beads out of adroplet.

“Washing” with respect to washing a bead means reducing the amountand/or concentration of one or more substances in contact with the beador exposed to the bead from a droplet in contact with the bead. Thereduction in the amount and/or concentration of the substance may bepartial, substantially complete, or even complete. The substance may beany of a wide variety of substances; examples include target substancesfor further analysis, and unwanted substances, such as components of asample, contaminants, and/or excess reagent. In some embodiments, awashing operation begins with a starting droplet in contact with amagnetically responsive bead, where the droplet includes an initialamount and initial concentration of a substance. The washing operationmay proceed using a variety of droplet operations. The washing operationmay yield a droplet including the magnetically responsive bead, wherethe droplet has a total amount and/or concentration of the substancewhich is less than the initial amount and/or concentration of thesubstance. Examples of suitable washing techniques are described inPamula et al., U.S. Pat. No. 7,439,014, entitled “Droplet-Based SurfaceModification and Washing,” issued on Oct. 21, 2008, the entiredisclosure of which is incorporated herein by reference.

The terms “top,” “bottom,” “over,” “under,” and “on” are used throughoutthe description with reference to the relative positions of componentsof the droplet actuator, such as relative positions of top and bottomsubstrates of the droplet actuator. It will be appreciated that thedroplet actuator is functional regardless of its orientation in space.

When a liquid in any form (e.g., a droplet or a continuous body, whethermoving or stationary) is described as being “on”, “at”, or “over” anelectrode, array, matrix or surface, such liquid could be either indirect contact with the electrode/array/matrix/surface, or could be incontact with one or more layers or films that are interposed between theliquid and the electrode/array/matrix/surface. In one example, fillerfluid can be considered as a film between such liquid and theelectrode/array/matrix/surface.

When a droplet is described as being “on” or “loaded on” a dropletactuator, it should be understood that the droplet is arranged on thedroplet actuator in a manner which facilitates using the dropletactuator to conduct one or more droplet operations on the droplet, thedroplet is arranged on the droplet actuator in a manner whichfacilitates sensing of a property of or a signal from the droplet,and/or the droplet has been subjected to a droplet operation on thedroplet actuator.

Barcodes and UMIs on Beads

In some embodiments, primers bearing sample barcodes can be immobilizedto a solid support. Additionally or alternatively, primers bearing UMIsequences can be immobilized to a solid support. For example, the solidsupport can be one or more beads. Thus, in certain embodiments, aplurality of beads can be presented, wherein each bead in the pluralitybears a unique sample barcode and/or UMI sequence. In some embodiments,individual cells are contacted with one or more beads having a uniqueset of sample barcodes and/or UMI sequences in order to identify theindividual cell. In some embodiments, lysates from individual cells arecontacted with one or more beads having a unique set of sample barcodesand/or UMI sequences in order to identify the individual cell lysates.In some embodiments, purified nucleic acid from individual cells arecontacted with one or more beads having a unique set of sample barcodesand/or UMI sequences in order to identify the purified nucleic acid fromthe individual cell. The beads can be manipulated in any suitable manneras is known in the art, for example, using droplet actuators asdescribed hereinabove.

The terms “solid surface,” “solid support” and other grammaticalequivalents herein refer to any material that is appropriate for or canbe modified to be appropriate for the attachment of the primers,barcodes and sequences described herein. As will be appreciated by thosein the art, the number of possible substrates is very large. Possiblesubstrates include, but are not limited to, glass and modified orfunctionalized glass, plastics (including acrylics, polystyrene andcopolymers of styrene and other materials, polypropylene, polyethylene,polybutylene, polyurethanes, Teflon™, etc.), polysaccharides, nylon ornitrocellulose, ceramics, resins, silica or silica-based materialsincluding silicon and modified silicon, carbon, metals, inorganicglasses, plastics, optical fiber bundles, and a variety of otherpolymers. Particularly useful solid supports and solid surfaces for someembodiments are located within a flow cell apparatus. Exemplary flowcells are set forth in further detail below.

In some embodiments, the solid support comprises a patterned surfacesuitable for immobilization of primers, barcodes and sequences describedherein in an ordered pattern. A “patterned surface” refers to anarrangement of different regions in or on an exposed layer of a solidsupport. For example, one or more of the regions can be features whereone or more transposome complexes are present. The features can beseparated by interstitial regions where transposome complexes are notpresent. In some embodiments, the pattern can be an x-y format offeatures that are in rows and columns. In some embodiments, the patterncan be a repeating arrangement of features and/or interstitial regions.In some embodiments, the pattern can be a random arrangement of featuresand/or interstitial regions. In some embodiments, the transposomecomplexes are randomly distributed upon the solid support. In someembodiments, the transposome complexes are distributed on a patternedsurface. Exemplary patterned surfaces that can be used in the methodsand compositions set forth herein are described in U.S. Ser. No.13/661,524 or US Pat. App. Publ. No. 2012/0316086 A1, each of which isincorporated herein by reference.

In some embodiments, the solid support comprises an array of wells ordepressions in a surface. This may be fabricated as is generally knownin the art using a variety of techniques, including, but not limited to,photolithography, stamping techniques, molding techniques andmicroetching techniques. As will be appreciated by those in the art, thetechnique used will depend on the composition and shape of the arraysubstrate.

The composition and geometry of the solid support can vary with its use.In some embodiments, the solid support is a planar structure such as aslide, chip, microchip and/or array. As such, the surface of a substratecan be in the form of a planar layer. In some embodiments, the solidsupport comprises one or more surfaces of a flowcell. The term“flowcell” as used herein refers to a chamber comprising a solid surfaceacross which one or more fluid reagents can be flowed. Examples offlowcells and related fluidic systems and detection platforms that canbe readily used in the methods of the present disclosure are described,for example, in Bentley et al., Nature 456:53-59 (2008), WO 04/018497;U.S. Pat. No. 7,057,026; WO 91/06678; WO 07/123744; U.S. Pat. Nos.7,329,492; 7,211,414; 7,315,019; 7,405,281, and US 2008/0108082, each ofwhich is incorporated herein by reference.

In some embodiments, the solid support or its surface is non-planar,such as the inner or outer surface of a tube or vessel. In someembodiments, the solid support comprises microspheres or beads. By“microspheres” or “beads” or “particles” or grammatical equivalentsherein is meant small discrete particles. Suitable bead compositionsinclude, but are not limited to, plastics, ceramics, glass, polystyrene,methylstyrene, acrylic polymers, paramagnetic materials, thoria sol,carbon graphite, titanium dioxide, latex or cross-linked dextrans suchas Sepharose, cellulose, nylon, cross-linked micelles and teflon, aswell as any other materials outlined herein for solid supports may allbe used. “Microsphere Detection Guide” from Bangs Laboratories, FishersInd. is a helpful guide. In certain embodiments, the microspheres aremagnetic microspheres or beads.

The beads need not be spherical; irregular particles may be used.Alternatively or additionally, the beads may be porous. The bead sizesrange from nanometers, i.e. 100 nm, to millimeters, i.e. 1 mm, withbeads from about 0.2 micron to about 200 microns being preferred, andfrom about 0.5 to about 5 micron being particularly preferred, althoughin some embodiments smaller or larger beads may be used.

Throughout this application various publications, patents and/or patentapplications have been referenced. The disclosure of these publicationsin their entireties is hereby incorporated by reference in thisapplication.

The term comprising is intended herein to be open-ended, including notonly the recited elements, but further encompassing any additionalelements.

A number of embodiments have been described. Nevertheless, it will beunderstood that various modifications may be made. Accordingly, otherembodiments are within the scope of the following claims.

What is claimed is:
 1. A method of preparing a cDNA library from aplurality of single cells comprising: releasing mRNA from each singlecell to provide a plurality of individual mRNA samples, wherein the mRNAin each individual mRNA sample is from a single cell; synthesizing afirst strand of cDNA from the mRNA in each individual mRNA sample with afirst strand synthesis primer mixture, wherein the first strandsynthesis primer mixture comprises a mixture of an oligo dT primer and arandomer primer, wherein the oligo dT primer and the randomer primereach comprise a tag to incorporate the tag into the cDNA to provide aplurality of tagged cDNA samples, wherein the cDNA in each tagged cDNAsample is complementary to mRNA from a single cell, and wherein each tagof each tagged cDNA sample has a same first portion comprising acell-specific identifier sequence, a same first-read sequencing adaptersequence, and a different second portion; pooling the tagged cDNAsamples generated from a plurality of single cells; amplifying thepooled, tagged cDNA samples to generate pooled, tagged, double-strandedcDNA; and performing a tagmentation reaction on the pooled, tagged,double-stranded cDNA to generate the cDNA library by contacting thepooled, tagged, double-stranded cDNA with a plurality of transposomecomplexes each comprising a transposase complexed with a transposoncomprising a transposon end sequence and a second-read sequencingadapter sequence, wherein the second-read sequencing adapter sequence isdifferent from the first-read sequencing adapter sequence and isidentical in all the transposons in the plurality of transposomecomplexes, wherein the transposome complexes used in the tagmentationreaction do not comprise the first-read sequencing adapter sequence, andwherein the cDNA library comprises a plurality of tagged cDNA fragmentshaving the first-read sequencing adapter sequence on a first strand andthe second-read sequencing adapter sequence on a second strand.
 2. Themethod of claim 1, further comprising amplifying the tagged cDNAfragments of the cDNA library to generate amplified, tagged cDNAfragments.
 3. The method of claim 2, wherein amplifying the tagged cDNAfragments comprises adding an additional sequence to the 5′ end of theamplification products.
 4. The method of claim 3, wherein the additionalsequence comprises a primer binding sequence for hybridization to acomplementary primer binding sequence on a solid support andamplification of the tagged cDNA fragments on the solid support.
 5. Themethod of claim 4, further comprising hybridizing the amplified, taggedcDNA fragments to the complementary primer binding sequence on the solidsupport, and amplifying the amplified, tagged cDNA fragments on thesolid support.
 6. The method of claim 5, further comprising sequencingthe amplification products on the solid support.
 7. The method of claim1, further comprising sequencing the tagged cDNA fragments of the cDNAlibrary.
 8. The method of claim 7, wherein sequencing comprises 3′ tagcounting.
 9. The method of claim 7, wherein sequencing comprises wholetranscriptome analysis.
 10. The method of claim 1, wherein the firststrand synthesis primer mixture comprises a primer comprising adouble-stranded portion.
 11. The method of claim 10, wherein the firststrand synthesis primer mixture reduces concatenation byproductscompared to a single-stranded first strand synthesis primer.
 12. Themethod of claim 10, wherein the first strand synthesis primer mixturecomprises a primer comprising a region capable of forming a hairpin. 13.The method of claim 10, wherein the first strand synthesis primermixture comprises a primer comprising a region of RNA.
 14. The method ofclaim 10, wherein the first strand synthesis primer mixture comprises aprimer hybridized to a complementary oligonucleotide, thereby forming adouble-stranded portion.
 15. The method of claim 1, wherein the tagcomprises a unique molecular identifier (UMI) sequence.
 16. The methodof claim 1, wherein the first strand synthesis primer mixture comprisesa primer attached to a bead and the synthesizing comprises synthesizingtagged cDNA samples on the bead.
 17. The method of claim 16, comprisingencapsulating each single cell in a droplet with the bead beforereleasing the mRNA from each single cell.
 18. The method of claim 17,wherein the releasing and synthesizing of each tagged cDNA sample isperformed in the droplet.
 19. The method of claim 16, wherein poolingthe tagged cDNA samples comprises pooling the beads with the attachedtagged cDNA samples.
 20. A method of preparing a cDNA library from aplurality of single cells comprising: releasing mRNA from each singlecell to provide a plurality of individual mRNA samples, wherein the mRNAin each individual mRNA sample is from a single cell; synthesizing afirst strand of cDNA from the mRNA in each individual mRNA sample with afirst strand synthesis primer and incorporating a tag into the cDNA toprovide a plurality of tagged cDNA samples, wherein the cDNA in eachtagged cDNA sample is complementary to mRNA from a single cell, andwherein the tag comprises a cell-specific identifier sequence, afirst-read sequencing adapter sequence, and a unique molecularidentifier (UMI) sequence; pooling the tagged cDNA samples generatedfrom a plurality of single cells; amplifying the pooled cDNA samples togenerate pooled, tagged, double-stranded cDNA; and performing atagmentation reaction on the pooled, tagged, double-stranded cDNA togenerate the cDNA library by contacting the pooled, tagged,double-stranded cDNA with a plurality of transposome complexes eachcomprising a transposase complexed with a transposon comprising atransposon end sequence and a second-read sequencing adapter sequencethat is identical in all the transposons in the plurality of transposomecomplexes, wherein the second-read sequencing adapter sequence isdifferent from the first-read sequencing adapter sequence, wherein thetransposome complexes used in the tagmentation reaction do not comprisethe first-read sequencing adapter sequence, and wherein the cDNA librarycomprises a plurality of tagged cDNA fragments having the first-readsequencing adapter sequence on a first strand and the second-readsequencing adapter sequence on a second strand.
 21. The method of claim20, further comprising amplifying the tagged cDNA fragments of the cDNAlibrary to generate amplified, tagged cDNA fragments.
 22. The method ofclaim 21, wherein amplifying the tagged cDNA fragments comprises addingan additional sequence to the 5′ end of the amplification products. 23.The method of claim 22, wherein the additional sequence comprises aprimer binding sequence for hybridization to a complementary primerbinding sequence on a solid support and amplification of the tagged cDNAfragments on the solid support.
 24. The method of claim 23, furthercomprising hybridizing the amplified, tagged cDNA fragments to thecomplementary primer binding sequence on the solid support, andamplifying the amplified, tagged cDNA fragments on the solid support.25. The method of claim 24, further comprising sequencing theamplification products on the solid support.
 26. The method of claim 20,further comprising sequencing the tagged cDNA fragments of the cDNAlibrary.
 27. The method of claim 26, wherein sequencing comprises 3′ tagcounting.
 28. The method of claim 20, wherein first strand synthesis isperformed using a mixture of random primers, wherein the random primerscomprise the tag, optionally wherein the first strand synthesis primercomprises a region capable of forming a hairpin.
 29. The method of claim20, wherein the first strand synthesis primer is hybridized to acomplementary oligonucleotide, thereby forming a double-strandedportion.
 30. A method of preparing a cDNA library from a plurality ofsingle cells comprising: releasing mRNA from each single cell to providea plurality of individual mRNA samples, wherein the mRNA in eachindividual mRNA sample is from a single cell; synthesizing a firststrand of cDNA from the mRNA in each individual mRNA sample with a firststrand synthesis primer and incorporating a tag into the cDNA to providea plurality of tagged cDNA samples, wherein the cDNA in each tagged cDNAsample is complementary to mRNA from a single cell, and wherein the tagcomprises a cell-specific identifier sequence and a unique molecularidentifier (UMI) sequence, and wherein the first strand synthesis primercomprises: (i) a mosaic end region having paired ends and positioned 5′of the UMI sequence, and (ii) a first-read sequencing adapter sequencein a hairpin region formed between the paired ends of the mosaic endregion; pooling the tagged cDNA samples generated from a plurality ofsingle cells; amplifying the pooled cDNA samples to generate pooled,tagged, double-stranded cDNA; and performing a tagmentation reaction onthe pooled, tagged, double-stranded cDNA to generate the cDNA library bycontacting the pooled, tagged, double-stranded cDNA with a plurality oftransposome complexes each comprising a transposase complexed with atransposon comprising a transposon end sequence and a second-readsequencing adapter sequence that is identical in all the transposons inthe plurality of transposome complexes, wherein the second-readsequencing adapter sequence is different from the first-read sequencingadapter sequence, wherein the transposome complexes used in thetagmentation reaction do not comprise the first-read sequencing adaptersequence, and wherein the cDNA library comprises a plurality of taggedcDNA fragments having the first-read sequencing adapter sequence on afirst strand and the second-read sequencing adapter sequence on a secondstrand.
 31. The method of claim 30, comprising amplifying the taggedcDNA fragments of the cDNA library to generate amplified, tagged cDNAfragments, wherein amplifying the tagged cDNA fragments comprises addinga primer binding sequence for hybridization to a complementary primerbinding sequence on a solid support to the 5′ end of the amplificationproducts.
 32. The method of claim 31, comprising: hybridizing theamplified, tagged cDNA fragments to a complementary primer bindingsequence on a solid support, amplifying the amplified, tagged cDNAfragments on the solid support to generate hybridized amplificationproducts, and sequencing the hybridized amplification products on thesolid support.
 33. The method of claim 20, wherein the first strandsynthesis primer is attached to a bead and the synthesizing comprisessynthesizing tagged cDNA samples on the bead.
 34. The method of claim33, comprising encapsulating each single cell in a droplet with the beadbefore releasing the mRNA from each single cell.
 35. The method of claim34, wherein the droplet is generated using a droplet actuator.
 36. Themethod of claim 34, wherein the releasing and synthesizing of eachtagged cDNA sample is performed in the droplet.
 37. The method of claim33, wherein pooling the tagged cDNA samples comprises pooling the beadswith the attached tagged cDNA samples.
 38. The method of claim 36,wherein pooling the tagged cDNA samples comprises pooling the beads withthe attached tagged cDNA samples.